14 A Practical Guide to Cancer Systems Biology
Figure 2. RNA electropherograms and the digital gel (right) obtained with the Agilent
2100 Bioanalyzer (L, ladder; C, control sample; T, treated sample; 100 ng per sample for
all RNAs). Total RNA was (a) extracted using Trizol and (b) subsequently treated with
DNase I to move genomic DNA contamination.
- Vortex the RPB tubes vigorously to completely resuspend the oligo-dT
beads. - Add 50μL of RPB to each sample to bind the poly-A RNA to the oligo-dT
magnetic beads. Gently pipette the entire volume up and down 10 times
to mix thoroughly. - Place the sample on the thermal cycler. Close the lid and denature the
RNA using the following program.
65 ◦Cfor5minutes, 4 ◦C hold- Remove the sample from the thermal cycler, centrifuge briefly, and allow
to stand on the bench for 5 minutes at room temperature to allow the
RNAtobindtothebeads. - Place the sample on the magnetic stand at room temperature for 5 minutes
to separate the poly-A RNA bound beads from the solution. - Remove and discard the supernatant from each sample.
- Remove the sample tube from the magnetic stand and wash the beads
with 200μl of BWB to remove unbound RNA. Gently pipette the entire
volume up and down 10 times to mix thoroughly. - Centrifuge briefly and place the sample on the magnetic stand at room
temperature for 5 minutes. - Remove and discard all of the supernatant from each sample.
Note: The supernatant contains the majority of the ribosomal RNA (rRNA) and other
non-mRNA.